Cloning,Prokaryotic Expression Of Colon Cancer Stem Cell Related Gene LYRM2 And Its Preparation Of Monoclonal Antibody

LYRM2(LYR motif containing 2) is located in 6q15 and conserved in human, chimpanzee, mouse,dog, chicken,rat, and zebrafish.This gene contains 4 transcript variants,only the transcript variant 1 can translate into protein,while others cannot initiate translation because of lacking the translational start codon. LYRM2 encodes 88 amino acids and belongs to the Complexl_LYR super family,which has been identified as a component of the higher eukaryotic NADH complex.However, the concret function of LYRM2 is still obscure.In order to study the role of LYRM2 in the colon cancer stem cells,it is imperative to prepare its antibody.In this study, we cloned the CDS of LYRM2 from colon cancer cell line SW480 by RT-PCR strategy. After the sequence confirmed,the segment was cloned into the vector(pET-43.1b) and under IPTG induction, LYRM2 recombinant protein about Mr 72000 was obtained. To obtain a large amount of soluble recombinant protein,we performed the fermentation of engineering bacteria. The soluble recombinant protein was then cut by enterokinase and purified by NI-NAT affinity column. The purifed protein was used as immunogen to immunize the nud mouse to produce monoclonal antibody,which lays an important experiment foundation for further studying; the function of LYRM2 in colon cancer stem cell.Objective:To clone LYRM2 gene, we construct prokaryotic expression vector of LYRM2 gene, purify LYRM2 protein,and prepare monoclonal antibody against LYRM2 and provide important experimental basis for further studying the function of LYRM2 in colon cancer stem cell.Methods: 1.The total RNA was extracted from colon cancer cell line SW480.According to information in GeneBank(NM₀20466.4), segment of CDS of LYRM2 was amplified by RT-PCR and was further cloned into the vector(pET-43.1b).2. After the sequence verified, the recombinant plasmid was transformed into E.coli.BLYRM21(DE3).The recombinant protein was expressed under IPTG induction and a large amount of soluble recombinant proteins were obtained through the fermentation of engineering bacteria,which were finally purified by NI affinity column.3. The recombinant protein was cut by enterokinase and purified by NI-NAT affinity column. The purifed protein was then used as an immunogen to immunize the nud mouse to produce monoclonal antibody, the mouse spleen cells were fused with mouse myeloma cell line SP2/02 Ag14, and the resulting hybridomas were screened for the production of LYRM2 specific antibodies by EL ISA assay and Western blot.Results:LYRM2 gene was amplified by RT-PCR from colon cancer cell line SW480 and an approximately 260bp band was shown as expected. via electrophoretogram.LYRM2 was further cloned into the vector (pET-43.1b), and the sequence was verified. The recombinant plasmid was transformed into E.coli.BLYRM21(DE3). A new protein band about Mr 72000 showed on SDS-PAGE through IPTG induction and coomassie brilliant blue dye. The recombinant protein was cut by enterokinase and purified by NI-NAT affinity column,the target protein band about Mr 15000 showed on SDS-PAGE through coomassie brilliant blue dye. The purifed target protein was then used as immunogen to immunize the nud mouse to produce monoclonal antibody, the mouse spleen cells were fused with mouse myeloma cell line SP2/02 Ag14.EL ISA assay and Western blot showed that the prepared monoclonal antibodies could specifically combined with LYRM2 and the brightness of target band varied from different antibody dilution.Conclusion:The prokaryotic expression vector pET-43.1b-LYRM2 was constructed successfully, the high purity target protein was obtained and monoclonal antibody was prepared. Western blot analyses showed that the monoclonal antibody could specifically recognized LYRM2 protein, which would facilitate the study of the function and modulation of LYRM2 in colon cance stem cells.